Microfluidic systems incorporating varied channel dimensions

ABSTRACT

The present invention is generally directed to improved microfluidic devices, systems and methods of using same, which incorporate channel profiles that impart significant benefits over previously described systems. In particular, the presently described devices and systems employ channels having, at least in part, depths that are varied over those which have been previously described. These varied channel depths provide numerous beneficial and unexpected results.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.08/948,194, filed Oct. 9, 1997 now U.S. Pat. No. 5,842,787.

BACKGROUND OF THE INVENTION

Microfluidic devices and systems have been gaining substantial interestas they are increasingly being demonstrated to be robust, highlyaccurate, high throughput and low cost methods of performing previouslycumbersome and or expensive analytical operations.

In particular, microfluidic systems have been described for use in ultrahigh throughput screening assay systems, e.g., for pharmaceuticaldiscovery, diagnostics and the like. See International Application No.PCT/US97/10894 filed Jun. 28, 1997 (Attorney Docket No. 17646-000420PC).In addition, such microfluidic systems have reportedly been used inperforming separations-based analyses, e.g., nucleic acid separations,etc. See, e.g., Woolley et al., Proc. Nat'l Acad. Sci., USA91:11348-11352 (1994).

Despite the promise of microfluidic systems in terms of throughput,automatability and cost, many of the systems that have been describedsuffer from substantial drawbacks. Initially, many of these systems havesubstantial reductions in resolution over their counterpart methods onthe bench top. In particular, a number of relatively minorconsiderations can readily become major factors when considered in thecontext of the relatively small amounts of material transported throughthese systems. For example, in microfluidic channels that include curvesor turns, variations in distances through these turns and curves at theinside and outside edges can substantially affect the resolution ofmaterials transported through these channels.

Further, simple operations, such as dilution and mixing have generallybeen accomplished at the expense of overall device volume, e.g., addingto the reagent/material volume required for carrying out the overallfunction of the device. In particular, such mixing typically requiresmuch larger chambers or channels in order to provide adequate mixing ofreagents or diluents within the confines f the microfluidic systems.

Thus, it would be generally desirable to provide microfluidic systemsthat are capable of capitalizing upon the myriad benefits describedabove, without sacrificing other attributes, such as resolution, volume,and the like. The present invention meets these and other needs.

SUMMARY OF THE INVENTION

The present invention generally provides microfluidic devices, systemsand methods of using these devices and systems. The microfluidic devicesand systems generally incorporate improved channel profiles that resultin substantial benefits over previously described microfluidic systems.

For example, in one embodiment, the present invention providesmicrofluidic devices and systems incorporating them, which devicescomprise a body structure and at least a first microscale channeldisposed therein. The microscale channel typically comprises at leastfirst and second ends and at least a portion of the microscale channelhaving an aspect ratio (width/depth) less than 1. In preferred aspects,the devices and systems include an electrical controller operably linkedto the first and second ends of the microscale channel, for applying avoltage gradient between the first and second ends, and/or arefabricated from polymeric materials.

In a related but alternate embodiment, the present invention providesmicrofluidic devices and systems that comprise a body structure havingat least a first microscale channel disposed therein, where themicroscale channel has at least one turning portion incorporatedtherein. In this embodiment, the turning portion of the channelcomprises a varied depth across its width, where the varied depth isshallower at an outside edge of the turning portion than at an insideedge of the turning portion. Preferably, the relative depths at theinside edge and outside edge of the turning portion of the channel areselected whereby the time required for a material traveling through theturning portion at the outside edge is substantially equivalent to atime required for the material to travel through the turning portion atthe inside edge.

As alluded to above, the present invention also comprises microfluidicsystems that include the above described microfluidic devices incombination with an electrical control system. The electrical controlsystem is operably coupled to the first and second ends of the first andsecond channels, and capable of concomitantly delivering a voltage toeach of the first and second ends of the first and second channels.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates a microfluidic device fabricated from aplanar substrate.

FIGS. 2A and 2B illustrate the distortion of material regions or plugswhen transported through a typical, curved microfluidic channel. FIG. 2Aillustrates distortion for a single material region, while FIG. 2Billustrates distortion for multiple separate species regions or bands,such as in electrophoretic separations analysis, as well as exemplarysignal, e.g., fluorescent signal that would be obtained from suchspecies bands, both before and after the distorting effects of thechannel curves.

FIG. 3 illustrates a diagram of an electric field applied across thelength of a turning microscale channel.

FIG. 4 illustrates a comparison of a channel having a shallow aspectratio, e.g., >1 (width/depth)(top), as well as a channel having a deepaspect ratio, e.g., <1 (width/depth)(bottom).

FIGS. 5A and 5B illustrate microscale channels having a varied depth topermit improved material intermixing.

DETAILED DESCRIPTION OF THE INVENTION

I. General

A. Introduction

The present invention is generally directed to improved microfluidicdevices, systems and methods of using same, which incorporate channelprofiles that impart significant benefits over previously describedsystems. In particular, the presently described devices and systemsemploy channels having, at least in part, depths that are varied overthose which have been previously described. These varied channel depthsprovide numerous beneficial and unexpected results.

As used herein, the term "microscale" or "microfabricated" generallyrefers to structural elements or features of a device which have atleast one fabricated dimension in the range of from about 0.1 μm toabout 500 μm. Thus, a device referred to as being microfabricated ormicroscale will include at least one structural element or featurehaving such a dimension. When used to describe a fluidic element, suchas a passage, chamber or conduit, the terms "microscale,""microfabricated" or "microfluidic" generally refer to one or more fluidpassages, chambers or conduits which have at least one internalcross-sectional dimension, e.g., depth, width, length, diameter, etc.,that is less than 500 μm, and typically between about 0.1 μm and about500 μm. In the devices of the present invention, the microscale channelsor chambers preferably have at least one cross-sectional dimensionbetween about 0.1 μm and 200 μm, more preferably between about 0.1 μmand 100 μm, and often between about 0.1 μm and 20 μm. Accordingly, themicrofluidic devices or systems prepared in accordance with the presentinvention typically include at least one microscale channel, usually atleast two intersecting microscale channels, and often, three or moreintersecting channels disposed within a single body structure. Channelintersections may exist in a number of formats, including crossintersections, "T" intersections, or any number of other structureswhereby two channels are in fluid communication.

The body structure of the microfluidic devices described hereintypically comprises an aggregation of two or more separate layers whichwhen appropriately mated or joined together, form the microfluidicdevice of the invention, e.g., containing the channels and/or chambersdescribed herein. Typically, the microfluidic devices described hereinwill comprise a top portion, a bottom portion, and an interior portion,wherein the interior portion substantially defines the channels andchambers of the device. For example, typically, the body structure isfabricated from at least two substrate layers that are mated together todefine the channel networks of the device, e.g., the interior portion.In preferred aspects, the bottom portion of the device comprises a solidsubstrate that is substantially planar in structure, and which has atleast one substantially flat upper surface.

A variety of substrate materials may be employed as the bottom portion.Typically, because the devices are microfabricated, substrate materialswill be selected based upon their compatibility with knownmicrofabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, LIGA, reactive ionetching (RIE), injection molding, embossing, and other techniques. Thesubstrate materials are also generally selected for their compatibilitywith the full range of conditions to which the microfluidic devices maybe exposed, including extremes of pH, temperature, salt concentration,and application of electric fields. Accordingly, in some preferredaspects, the substrate material may include materials normallyassociated with the semiconductor industry in which suchmicrofabrication techniques are regularly employed, including, e.g.,silica based substrates, such as glass, quartz, silicon or polysilicon,as well as other substrate materials, such as gallium arsenide and thelike. In the case of semiconductive materials, it will often bedesirable to provide an insulating coating or layer, e.g., siliconoxide, over the substrate material, and particularly in thoseapplications where electric fields are to be applied to the device orits contents.

In particularly preferred aspects, the substrate materials will comprisepolymeric materials, e.g., plastics, such as polymethylmethacrylate(PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™),polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, andthe like. Such polymeric substrates are readily manufactured usingavailable microfabrication techniques, as described above, or frommicrofabricated masters, using well known molding techniques, such asinjection molding, embossing or stamping, or by polymerizing thepolymeric precursor material within the mold (See U.S. Pat. No.5,512,131). Such polymeric substrate materials are preferred for theirease of manufacture, low cost and disposability, as well as theirgeneral inertness to most extreme reaction conditions. Again, thesepolymeric materials may include treated surfaces, e.g., derivatized orcoated surfaces, to enhance their utility in the microfluidic system,e.g., provide enhanced fluid direction, e.g., as described in U.S.patent application Ser. No. 08/843,212, filed Apr. 14, 1997, and whichis incorporated herein by reference in its entirety for all purposes.

An example of a microfluidic device fabricated from a planar substrateis illustrated in FIG. 1. Briefly, the channels and/or chambers of thedevice 100 are typically fabricated into or upon a flat surface of aplanar substrate 102. The channels and/or chambers of the device may befabricated using a variety of methods whereby these channels andchambers are defined between two opposing substrates. For example, thechannels, e.g., channels 104 and 106, and/or chambers of themicrofluidic devices are typically fabricated into the upper surface ofthe bottom substrate or portion, as microscale grooves or indentations,using the above described microfabrication techniques. Alternatively,raised regions may be fabricated onto the planar surface of the bottomportion or substrate in order to define the channels and/or chambers.The top portion or substrate (not separately shown) also comprises afirst planar surface, and a second surface opposite the first planarsurface. In the microfluidic devices prepared in accordance with themethods described herein, the top portion also includes a plurality ofapertures, holes or ports 110-116 disposed therethrough, e.g., from thefirst planar surface to the second surface opposite the first planarsurface.

The first planar surface of the top substrate is then mated, e.g.,placed into contact with, and bonded to the planar surface of the bottomsubstrate, covering and sealing the grooves and/or indentations in thesurface of the bottom substrate, to form the channels and/or chambers(i.e., the interior portion) of the device at the interface of these twocomponents. The holes 110-116 in the top portion of the device areoriented such that they are in communication with at least one of thechannels, e.g., 104 or 106, and/or chambers formed in the interiorportion of the device from the grooves or indentations in the bottomsubstrate. In the completed device, these holes 110-116 function asreservoirs for facilitating fluid or material introduction into thechannels or chambers of the interior portion of the device, as well asproviding ports at which electrodes may be placed into contact withfluids within the device, allowing application of electric fields alongthe channels of the device to control and direct fluid transport withinthe device. As shown, channel 104, which serves as the main or analysischannel in the device shown, intersects channel 106, which serves as asample introduction channel, at intersection 108. The analysis channel104, also includes a serpentine portion 118, which serves to extend thelength of the analysis channel without requiring substantially greatersubstrate area.

In many embodiments, the microfluidic devices will include an opticaldetection window 120 disposed across one or more channels and/orchambers of the device. Optical detection windows are typicallytransparent such that they are capable of transmitting an optical signalfrom the channel/chamber over which they are disposed. Optical detectionwindows may merely be a region of a transparent cover layer, e.g., wherethe cover layer is glass or quartz, or a transparent polymer material,e.g., PMMA polycarbonate, etc. Alternatively, where opaque substratesare used in manufacturing the devices, transparent detection windowsfabricated from the above materials may be separately manufactured intothe device.

These devices may be used in a variety of applications, including, e.g.,the performance of high throughput screening assays in drug discovery,immunoassays, diagnostics, nucleic acid analysis, including geneticanalysis, and the like. As such, the devices described herein, willoften include multiple sample introduction ports or reservoirs, for theparallel or serial introduction and analysis of multiple samples, e.g.,as described in U.S. patent application Ser. No. 08/845,754, filed Apr.25, 1997, and incorporated herein by reference. Alternatively, thesedevices may be coupled to a sample introduction port, e.g., a pipettor,which serially introduces multiple samples into the device for analysis.Examples of such sample introduction systems are described in e.g., U.S.patent application Ser. Nos. 08/761,575 and 08/760,446 (Attorney DocketNos. 17646-000410 and 17646-000510, respectively) each of which wasfiled on Dec. 6, 1996, and is hereby incorporated by reference in itsentirety for all purposes.

II. Channel Aspect Ratios

In a first aspect, the present invention provides microfluidic devicesand systems that comprise a body structure which has disposed therein,at least one microscale channel or channel portion, which channel orchannel portion has an aspect ratio that is substantially the inverse ofpreviously described microscale channels.

In particular, microscale channels which have been described for use inmicrofluidic systems, typically have employed channel dimensions in therange of from about 50 to about 200 μm wide and from about 5 to about 20μm deep. In any event, the aspect ratios of these channels (width/depth)is well in excess of 2 and typically is in the range of from about 7 toabout 10. These aspect ratios have likely resulted, at least in part,from the processes involved in the fabrication of microfluidic systems,and particularly, the microscale channels incorporated therein.Specifically, such channels are often fabricated in silicon basedsubstrates, such as glass, quartz, silicon, etc., usingphotolithographic techniques. The chemistries involved in suchtechniques are readily used to fabricate channels having widths anddepths in the above-described ranges. However, because these techniquesinvolve etching processes, i.e., using generally available isotropicetching chemicals e.g., hydrofluoric acid (HF), they are generally notas effective in producing channels having depths substantially greaterthan those described, due to the increased etching time required.Specifically, isotropic agents typically etch uniformly in alldirections on amorphous substrates, i.e., glass. In such instances, itbecomes effectively impossible to produce channels having aspect ratiosless than 1.

Microfluidic devices incorporating the above-described dimensions andaspect ratios, have proven very useful in a wide variety of importantanalytical applications. These applications include high-throughputscreening of pharmaceutical and other test compounds (See commonlyassigned U.S. application Ser. No. 08/761,575, filed Dec. 6, 1996),nucleic acid analysis, manipulation and separation (See commonlyassigned U.S. application Ser. Nos. 08/835,101 and 08/845,754, filedApr. 4, 1997 and Apr. 25, 1997, respectively), and more.

Despite the substantial utility of these systems, the present inventorshave identified some potential shortcomings of microscale channelshaving the above-described dimensions, particularly where it is desiredto transport a given material region over a substantial distance withinthese channels.

In a first particular example, because microfluidic systems aretypically fabricated within body structures that have relatively smallareas, it is generally desirable to maximize the use of the space withinthe body structure. As such, channels often incorporate geometries thatinclude a number of channel turns or corners, e.g., serpentine,saw-tooth etc. The incorporation of such channel turns can have adverseeffects on the ability of discrete material regions to maintain theircohesion. In particular, in a turning microscale channel, materialtravelling along the outside edge traverses the turn much more slowlythan material travelling at the inside edge, imitating a "race-track"effect. This effect is at least in part due to the greater distance, orin the case of a three dimension fluidic system, the greater volume amaterial must travel through at the outside edge of a channel as opposedto the inner edge of the channel. This difference in traversal time canresult in a substantial perturbation or distortion of a discrete,cohesive material plug or region in a microscale channel.

A schematic illustration of this sample perturbation resulting fromturns or curves in microscale channels is provided in FIG. 2A. Briefly,FIG. 2A illustrates a discrete material region 204, e.g., a sample plug,species band or the like, travelling through a microscale channel 202.In the straight portions of the channel, the material regionsubstantially maintains its shape with a certain level of diffusion.However, once the material region travels around a curve in the channel,the differences in flow rate through the channel at different pointsacross the channel's width, result in a distorted material region 206.

The distortion of these material regions can adversely effect theresolution with which the particular material region is transportedthrough the turning channel. This is particularly problematic, forexample, in separation applications, e.g., protein or nucleic acidelectrophoresis, where the goal is to separate a given material into itsconstituent elements, and to separately detect those elements. Further,such separation applications typically require substantially longerseparation channels or columns, thereby increasing the number of channelturns to which a particular material will be subjected. This separatedetection typically requires that the elements be maintained as wellresolved material regions. FIG. 2B illustrates an exemplary separationchannel incorporating a channel turn. The separated species bands 214substantially maintain their shape and separation within the straightportions of channel 202. The well-resolved character of the speciesbands 214 is illustrated by the signal graph 216. After having traveledthrough the curved section of the channel, the species bands 218 showsubstantial distortion. Further, as indicated by the signal graph 220,the resolution of these bands, and thus their detectability, issubstantially reduced.

This material region distortion, or sample perturbation, also becomes aproblem where one wishes to separately transport materials through amicrofluidic system, e.g., for separate analysis. Examples of suchapplications include high-throughput experimentation, e.g., screening,diagnostics, and the like. In particular, maintaining differentmaterials, e.g., samples, as well-resolved plugs of material, i.e.,without the above-described distortion effects, allows multiple plugs tobe moved through a system without fear of intermixing. This alsoprevents the excess, uncontrolled dilution of those samples, resultingfrom the distortion effects.

In certain preferred aspects, the microfluidic devices and systemsdescribed herein, employ electrokinetic material transport systems formoving and directing material through and among the microscale channelnetworks that are incorporated in the microfluidic devices.Unfortunately, however, the level of sample perturbation issubstantially increased in microfluidic systems that employ suchelectrokinetic material transport.

As used herein, "electrokinetic material transport systems" includesystems which transport and direct materials within an interconnectedchannel and/or chamber containing structure, through the application ofelectrical fields to the materials, thereby causing material movementthrough and among the channel and/or chambers, i.e., cations will movetoward the negative electrode, while anions will move toward thepositive electrode.

Such electrokinetic material transport and direction systems includethose systems that rely upon the electrophoretic mobility of chargedspecies within the electric field applied to the structure. Such systemsare more particularly referred to as electrophoretic material transportsystems. Other electrokinetic material direction and transport systemsrely upon the electroosmotic flow of fluid and material within a channelor chamber structure which results from the application of an electricfield across such structures. In brief, when a fluid is placed into achannel which has a surface bearing charged functional groups, e.g.,hydroxyl groups in etched glass channels or glass microcapillaries,those groups can ionize. In the case of hydroxyl functional groups, thisionization, e.g., at neutral pH, results in the release of protons fromthe surface and into the fluid, creating a concentration of protons atnear the fluid/surface interface, or a positively charged sheathsurrounding the bulk fluid in the channel. Application of a voltagegradient across the length of the channel, will cause the proton sheathto move in the direction of the voltage drop, i.e., toward the negativeelectrode.

"Controlled electrokinetic material transport and direction," as usedherein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. Controlled electrokinetic material transport isdescribed in Published PCT Application No. WO 96/04547, to Ramsey, whichis incorporated herein by reference in its entirety for all purposes. Inparticular, the preferred microfluidic devices and systems describedherein, include a body structure which includes at least twointersecting channels or fluid conduits, e.g., interconnected, enclosedchambers, which channels include at least three unintersected termini.The intersection of two channels refers to a point at which two or morechannels are in fluid communication with each other, and encompasses "T"intersections, cross intersections, "wagon wheel" intersections ofmultiple channels, or any other channel geometry where two or morechannels are in such fluid communication. An unintersected terminus of achannel is a point at which a channel terminates not as a result of thatchannel's intersection with another channel, e.g., a "T" intersection.In preferred aspects, the devices will include at least threeintersecting channels having at least four unintersected termini. In abasic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic material flow of this materialacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage). However, this type of materialflow through the intersection would result in a substantial amount ofdiffusion at the intersection, resulting from both the natural diffusiveproperties of the material being transported in the medium used, as wellas convective effects at the intersection.

In controlled electrokinetic material transport, the material beingtransported across the intersection is constrained by low level flowfrom the side channels, e.g., the top and bottom channels. This isaccomplished by applying a slight voltage gradient along the path ofmaterial flow, e.g., from the top or bottom termini of the verticalchannel, toward the right terminus. The result is a "pinching" of thematerial flow at the intersection, which prevents the diffusion of thematerial into the vertical channel. The pinched volume of material atthe intersection may then be injected into the vertical channel byapplying a voltage gradient across the length of the vertical channel,i.e., from the top terminus to the bottom terminus. In order to avoidany bleeding over of material from the horizontal channel during thisinjection, a low level of flow is directed back into the side channels,resulting in a "pull back" of the material from the intersection.

In addition to pinched injection schemes, controlled electrokineticmaterial transport is readily utilized to create virtual valves whichinclude no mechanical or moving parts. Specifically, with reference tothe cross intersection described above, flow of material from onechannel segment to another, e.g., the left arm to the right arm of thehorizontal channel, can be efficiently regulated, stopped andreinitiated, by a controlled flow from the vertical channel, e.g., fromthe bottom arm to the top arm of the vertical channel. Specifically, inthe `off` mode, the material is transported from the left arm, throughthe intersection and into the top arm by applying a voltage gradientacross the left and top termini. A constraining flow is directed fromthe bottom arm to the top arm by applying a similar voltage gradientalong this path (from the bottom terminus to the top terminus). Meteredamounts of material are then dispensed from the left arm into the rightarm of the horizontal channel by switching the applied voltage gradientfrom left to top, to left to right. The amount of time and the voltagegradient applied dictates the amount of material that will be dispensedin this manner.

Although described for the purposes of illustration with respect to afour way, cross intersection, these controlled electrokinetic materialtransport systems can be readily adapted for more complex interconnectedchannel networks, e.g., arrays of interconnected parallel channels.

In order to provide the necessary voltage gradients to the various portsor reservoirs of a microfluidic device, e.g., as described above, themicrofluidic systems generally include electrical controllers whichinclude a plurality of electrical leads that are operably coupled to theseparate ports of the microfluidic device, e.g., in electricalconnection. The electrical controller is then capable of separatelydelivering a voltage to each of the different leads and ports. Examplesof a particularly preferred electrical controller is described in, e.g.,International Patent Application No. PCT/US97/12930, filed Jul. 3, 1997,and incorporated herein by reference in its entirety.

As noted above, in microfluidic systems employing electrokineticmaterial transport, sample perturbation around channel turns can besomewhat exacerbated. In particular, the causes of the differential rateat which materials at the inside and outside edges travel around theturn are two-fold. Initially, materials at the outside edge, asdescribed above, must travel farther than materials at the inside edgeof a turning channel and thus will take longer to traverse a turn. Inaddition and perhaps more importantly, under electrokinetic materialtransport, the movement of material is dictated by the nature of theelectric field being applied across the region through which thatmaterial travels. Specifically, materials move in relation to the amountand direction of electric current. The nature of this electricfield/current is substantially different at the inner edge of theturning channel than at the outer edge of the channel. A schematicillustration of the electric field that exists at such channel turns isshown in FIG. 3. In brief, within curving or turning channel 102, theelectric field, e.g., as applied by negative and positive electrodes 304and 306, respectively, is much more concentrated at the inner edge ofthe channel versus the outer edge of the channel, as shown by theconstant voltage lines 302 across the channel. The electric field ishigher where the voltage lines are closer together

The differences at the inner and outer edges of the channel can bereduced by making the turning channel narrower, and thus reducing thedifference in the radii of these two edges. By reducing the differencein the radii of these two edges, one proportionally reduces thedifference in volume through which materials at these two edges musttravel. However, this solution also requires a narrowing of the channelaround the corner, resulting in increased resistance through thechannel, lower throughput, and all the associated consequences, i.e.,increased current heating, higher pressures, etc.

The present invention addresses the difficulties associated with theproblems of turning channels, by providing channels that are narrower inwidth, thereby reducing differences in the volume through which thematerials must travel at the inner and outer edges of a channel, butwhich channels are substantially deeper, thereby preventing increases inresistance or pressure. Specifically, and as noted above, the presentinvention provides microfluidic devices and systems having microscalechannels, wherein at least a portion of the channel or channels isdeeper and narrower than previously described, in order to mitigate someof the problems that have been encountered in microfluidics. Inaddition, by providing these channels as deeper regions, the differencein volumes through which material must travel at the inner and outeredges, becomes a smaller percentage of the overall volume of the channelaround the curve, and thus has a smaller effect on the differential flowrate of material travelling at these different edges.

In particular, the present invention provides microfluidic devices andsystems wherein one or more microscale channels contained within thedevices and systems include channel regions having an aspect ratio ofless than 1. As used herein, the term "aspect ratio," refers to theratio of a channel's width to that channel's depth. Thus, a channelhaving an aspect ratio of less than 1, is deeper than it is wide, whilea channel having an aspect ratio greater than 1 is wider than it isdeep.

Typically, the channel portions described herein, will be in the rangeof from about 5 to about 50 μm wide, while being from about 20 to about200 μm deep. In preferred aspects, the aspect ratio of these channelportions will be less than or equal to about 0.5, e.g., more than twiceas deep as it is wide, more preferably, about 0.2, and often in therange of about 0.1 or less. A comparison of shallow aspect ratiochannels, e.g., those having an aspect ratio greater than 1(width/depth) is shown in FIG. 4 (top), where the channel 406 is shownfrom an end-on perspective, fabricated into a lower substrate 404, whichhas been mated or bonded with upper substrate 402, to define thechannel. In the bottom panel, a channel 408 is illustrated from the sameperspective, but where the channel has an aspect ratio less than 1(width/depth). As shown, both channels 406 and 408 have the samecross-sectional area, so as not to affect the level of material flow orcurrent flow (resistance) through the channel.

Fabrication of microfluidic systems that incorporate the aspect ratiosdescribed herein, may generally be carried out using the above-describedmicrofabrication methods. For example, crystalline substrates may beused that etch nonisotropically, and in which channels havingappropriate aspect ratios are fabricated. Preferably, however, themicrofluidic systems described herein are fabricated in polymericsubstrates, as described above. Fabrication techniques used withpolymeric substrates, e.g., injection molding, embossing, laser ablationand the like, permit the more rapid formation of substrates withappropriate depth dimensions, e.g., using LIGA methods in combinationwith computer aided design (CAD). Further, in the case of moldingmethods, the molds used to fabricate these polymeric substrates aregenerally fabricated from materials that are more easily manufactured,e.g., crystalline substrates, such as silicon or photoresists,permitting the use of methods capable of producing much deeper aspectratios, e.g., LIGA methods. For example, etching positive molds using UVor x-ray etching in photoresist, or reactive ion etching directly insolid substrates such as glass or silicon, permits the production ofchannels having deep, straight etch profiles. These molds are then usedto create negative electroforms, e.g., nickel electroforms, which inturn, are used to cast or injection mold polymeric substrates. Usingsuch methods, aspect ratios of less than 0.02 (width/depth) are readilyrealized. In the case of forming polymeric substrates for use inaccordance with the present invention, aspect ratios of 0.1, 0.2, 0.25,0.3 and the like are readily achieved. Further, masters incorporatingnegative structures having these aspect ratios, e.g., raised ridgescorresponding to the channels of the eventual device/substrate,typically provide more durable masters for use in the injection moldingor casting processes.

In addition to the foregoing advantages, bonding of substrate layersalso is facilitated in channels having aspect ratios described herein.In particular, in polymeric substrate based microfluidic devicesincorporating previous aspect ratios, e.g., from about 2 to about 10,the upper interior wall surface on a given channel provided asubstantial percentage of the overall interior surface of the channel,e.g., from about 1/3 to about 1/2 of the overall interior surface.Variations of this upper surface resulting from the bonding process havesubstantial adverse effects upon the nature of material transport withinthe channel. Such variations include the presence of adhesives used inthe bonding process, on the upper interior surface, or in the case ofthermal or solvent bonding processes, melting of the upper surface. Thismelting can cause variations or distortions within the upper surface, orto the channel shape, at or near the upper surface. All of thesevariations can potentially cause distorted flow characteristics in amicrofluidic channel.

In accordance with the present invention, this upper interior surfacemakes up a substantially reduced proportion of the overall interiorsurface of the channel, e.g., less than 1/4 of the overall interiorsurface. As such, any adverse effects resulting therefrom are alsosubstantially reduced.

As noted above, the microfluidic devices described herein, e.g., havingaspect ratios less than 1, are particularly useful in those deviceswhich include channels having one, two, three, four, five, six or morecorners, turns, etc. As used herein, the term "turn" or "corner" whenreferring to a channel geometry, refers to a channel having a directionchange of at least 30°. Further, the term "turn" does not necessarilyrequire an abrupt direction change, but also encompasses curved or arcedchannel turns. Typically, such turns or corners will be at least 45°,while preferred microfluidic devices will include channels thatincorporate turns of greater than 90° and often greater than 150°.Often, as is the case of serpentine channel structures, a single channelwill incorporate several turns, e.g., 2, 3, 4 or more, that are as largeas 180°.

The microfluidic devices described herein may include the aspect ratiosdescribed, e.g., less than 1, with respect to all channels containedwithin a particular device, e.g., one or more intersecting channels,some of these channels, a single channel and even a portion or portionsof one or more channels. For example, in channel structures thatincorporate turns, only that portion of the channel at the turn mayinclude such an aspect ratio. Specifically, channels may be providedhaving a more typical aspect ratio, e.g., greater than 2, in thoseportions where the channel is straight. However, at the turns, theaspect ratio is varied to less than 1. Preferably, however, the entirechannel will have an aspect ratio of less than 1.

As noted above, the problems of sample perturbation are particularlytroubling in those applications where one is transporting a materialthrough relatively long turning channels, and particularly where one isdesirous of maintaining that material as a well resolved region, plug orspecies band, as it travels through that system. Accordingly, themicrofluidic devices and systems described herein, e.g., having achannel, which includes at least a channel portion having an aspectratio of less than about 1, are particularly useful in microfluidicsystems that employ such elongated channels. Such elongated channels areparticularly useful in separation based analyses, e.g., separating amixture of components into several discrete species bands, such asnucleic acid separations, protein separations, etc., where longerchannels provide greater separation, e.g., greater numbers oftheoretical plates.

Thus, in at least one embodiment, the microfluidic devices and systemsdescribed herein include a separation channel having an aspect ratiothat is less than 1. In particular, separation channels typically gainresolution with increasing length. As such, these separation channelstypically include one or more turns, and preferably include a repeatingserpentine geometry. Typically, such separation channels will alsoinclude a separation matrix disposed therein. Separation matricessuitable for microfluidic systems are generally commercially available,i.e., GeneScan™ polymers available from Applied Biosystems/Perkin-Elmer,and the like.

The separation channel may be the main or only channel within themicrofluidic device. However, in preferred aspects, the separationchannel is in fluid communication with, e.g., intersects, at least oneadditional channel disposed within the microfluidic system, typically asample introduction channel. In preferred aspects, a number of distinctsample introduction channels are provided in fluid communication withthe separation channel, thus allowing analysis of multiple sampleswithin a single device. Examples of particularly preferred channelgeometries for carrying out separation based analyses are described incommonly assigned and co-pending U.S. application Ser. No. 08/845,754,filed Apr. 25, 1997 (Attorney Docket No. 100/01000), incorporated hereinby reference in its entirety for all purposes.

III. Mixing/Dilution in Microscale Channels

A second example of problems encountered in microfluidic systems relatesto the intermixing of different materials within microscale channelstructures. In most instances, mixing of different materials, fluids orspecies, e.g., from two or more different sources, in a microfluidicsystem, is typically carried out by diffusional mixing of thosematerials within a microscale channel. Specifically, where it is desiredto mix two materials, e.g., fluids, to perform a given analysis,synthesis, etc., each of the two materials is introduced, simultaneouslyinto a microscale channel. However, even at the extremely smalldimensions of these microscale channels, substantially completediffusional mixing of discretely introduced materials requires a certainamount of time, which can vary depending upon the medium, the molecularsize of the materials, e.g., the diffusion constants, and the like.Because reactions within microfluidic systems often are carried out "onthe fly," within flowing channels, the channels in which these materialsare to be mixed are generally provided having a sufficient length, inorder to provide the materials with adequate time for substantiallycomplete diffusional mixing. This results in channel structures thatdevote a large amount of channel length, and thus substrate area, tomixing applications. As used herein, the phrase "substantially completediffusional mixing" refers to the mixing of two or more materials bydiffusion, to form a substantially homogenous mixture of thesematerials.

The present invention, addresses this problem of microfluidic systems byproviding deeper channel regions within the system, to permit adequatediffusional mixing within a shorter channel length. In particular, ittakes longer for a given volume of material to travel through a deeperchannel, assuming the same volume flow rate. This provides more time formaterials to mix. In addition, and perhaps more importantly, thediffusion characteristics within these deeper channels are substantiallyenhanced. This is evident whether one introduces two or more volumes ofmaterial, serially, or in parallel into the mixing channel.Specifically, because diffusional mixing of two adjacent materialregions, e.g., in a channel, is largely dictated by lateral diffusion ofthe material between two material regions, by providing those regions asthinner layers, either in a serial or parallel orientation, one cansubstantially enhance the rate at which the two layers willsubstantially completely diffuse together.

The enhanced mixing of serially introduced materials within deeperchannels is schematically illustrated in FIG. 5A. In particular, twomaterials 504 and 506 serially introduced into a shallow channel 502will diffuse at a diffusion rate that depends upon the viscosity of themedium, the molecular size of the materials, etc., as indicated byarrows 508 and 510. In a deeper channel portion 512, however, the samediffusional kinetics, as shown by arrows 508 and 510, result in morecomplete diffusional mixing at a much faster rate, as the material mustdiffuse a much shorter distance, and has a greater interfacial areaacross which to diffuse. While similar advantages are also gained insuch serial systems, by providing a wide mixing channel (as opposed to adeep channel), one utilizes a substantially greater amount of substratearea.

By providing deeper and narrower mixing channels, one enhances thediffusional mixing of materials, without increasing the amount ofhydrodynamic flow relative to the electroosmotic flow, or altering thelevel of electrical resistance through that channel. In particular, thearea along which the adjacent material regions interface in the mixingchannel is substantially increased relative to the thickness of eachregion. This permits the more efficient intermixing, as described.However, by maintaining at least one thin dimension within the mixingchannel (typically width), or within all other channels of themicrofluidic device, there is little or no increase in hydrodynamic floweffects within these channels. Alternatively, mixing channels can benarrowed to provide enhanced mixing kinetics from materials beingtransported in on either side of the channel. In such instances,providing these mixing channels as deeper channel portions permits suchnarrowing, without increasing the level of resistance across the lengthof that mixing channel portion.

FIG. 5B provides schematic illustration of a deep mixing channel, e.g.,having an aspect ratio as described herein, where materials that are tobe mixed are introduced in parallel. As shown in the top panel, channels514 and 516 intersect in main channel 518. The materials to be mixed,504 and 506, are concomitantly introduced into main channel 518 fromchannels 514 and 516, respectively. Again, the diffusion kinetics of thematerials are illustrated by arrows 508 and 510. As illustrated in thebottom panel, channels 514 and 516 intersect a narrower and deeperchannel portion 520. In this narrower/deeper channel portion 520,materials 504 and 506 mix much more quickly as a result of the samediffusion kinetics (arrows 508 and 510). Typically, this deeper/narrowerchannel portion will have an aspect ratio as described above, e.g., lessthan 1 (width/depth). By providing at least the mixing channel portionwith an aspect ratio in accordance with the present invention, oneachieves not only the advantages of reducing the distance molecules mustdiffuse, but also maintains a suitable cross-sectional area of thechannel, e.g., as compared with typical channels having aspect ratiosmuch greater than 1. This allows a narrowing of the mixing channel whileallowing control or maintenance of the electrical resistance of thechannel, as described above.

In addition to the above described advantages of enhanced mixing,unaltered channel resistance, use of less substrate area, and the like,such narrower deeper channels also provide for the same level ofresolution as channels having the inverse aspect ratio. In particular,simply narrowing a channel to achieve enhanced mixing would result in aspreading of discrete species bands or plugs disposed in that channel,resulting in lower resolution detection of those bands. However, becausethe channels described herein are deeper, in addition to being narrower,the species bands do not spread, and no loss of resolution is observed.Further, detection optics in microfluidic systems typically look through(perpendicular to) the plane of the substrate in order to detect signalsin a given channel. In doing so, these optics are typically directed ata portion of the channel in which detection is desired. In the case ofmicrofluidic devices incorporating channel aspect ratios describedherein, however, such deeper narrower channels permit the analysis bythe detection optics, of greater numbers of detectable molecules, e.g.,labeling groups, by looking through the deeper, but narrower dimensionof the channel.

As noted, the improved mixing kinetics created by the devices of presentinvention, provide substantially improved mixing in smaller substrateareas. Further, such improved mixing permits a time-based approach tomixing and dilution, as opposed to a volume-based approach.Specifically, in previous methods, mixing or dilution of one materialwith another is carried out by delivering different volumes of thematerials to a common chamber or channel, simultaneously, in order toallow maximal mixing of these materials. In the microfluidic devices ofthe present invention, optimal mixing is provided by increasing themixing channels depth. As such, materials can be combined at apreselected ratio, by transporting each material into the mixingchannel, serially, and at full strength. The relative ratio of onematerial to the other is dictated by the amount of time each material istransported into the mixing channel, and the flow rate at which eachmaterial is transported into that channel. For example, assuming eachmaterial has an equivalent flow rate, the concentration ratio of onematerial to the other is directly proportional to the time each materialwas pumped into the common channel. Because these mixing channels haveenhanced mixing characteristics, the serially introduced materials arecapable of substantially completely mixing, in shorter channel lengths.

The dimensions of the mixing channel will typically vary depending uponthe nature of the materials to be mixed. Specifically, larger moleculesare slower to diffuse, and thus require a mixing channel that has eithera greater length, or a greater cross-sectional area, such that thematerials are substantially mixed while they are within the mixingchannel. Typically, the dimensions of a mixing channel will fall withinthe range of dimensions for the microscale channels, described above.However, such channels will typically have an aspect ratio of less than1, and preferably, less than or equal to 0.5, e.g., more than twice asdeep as wide, more preferably, less than or equal to about 0.3, stillmore preferably, less than or equal to about 0.25, or less than or equalto about 0.2, and often in the range of about 0.1 to 0.05. In preferredinstances, these mixing channel portions will have dimensions in therange of from about 2 to about 50 μm wide, while being from about 10 toabout 200 μm deep. In particularly referred aspects, the mixing channelswill be from about 5 to about 20 μm wide, and from about 50 to about 200μm deep. While mixing channels can incorporate wide regions as opposedto deep regions, such wider regions occupy greater amounts of substratearea, and as such, are less preferred. In any event, mixing channelportions will typically range from about 0.1 to about 10 mm in length,and are preferably in the range of from about 0.1 to about 3 mm inlength

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity and understanding,it will be apparent that certain changes and modifications may bepracticed within the scope of the appended claims. All publications,patents and patent applications referenced herein are herebyincorporated by reference in their entirety for all purposes as if eachsuch publication, patent or patent application had been individuallyindicated to be incorporated by reference.

What is claimed is:
 1. A microfluidic device, comprising:a firstsubstrate layer, having at least a first groove fabricated into a firstplanar surface thereof; a second substrate layer having a first planarsurface, the first planar surface of the second substrate layer beingbonded to the first planar surface of the first substrate layer to sealthe groove and define a first channel between the first and secondsubstrate layers, the first planar surface of the second substrate layerdefining a top inner surface of the first channel, the top inner surfaceof the first channel comprising 1/4 or less of a total internal surfacearea of the first channel.
 2. The microfluidic device of claim 1,wherein the first groove is from about 20 μm to about 200 μm deep. 3.The microfluidic device of claim 1, wherein the first groove is fromabout 5 μm to about 50 μm wide.
 4. The microfluidic device of claim 1,wherein the first surface of the first substrate layer is thermallybonded to the first surface of the second substrate layer.
 5. Themicrofluidic device of claim 1, wherein the first surface of the firstsubstrate layer is solvent bonded to the first surface of the secondsubstrate layer.
 6. The microfluidic device of claim 1, wherein thefirst surface of the first substrate layer is adhesive bonded to thefirst surface of the second substrate layer.
 7. The microfluidic deviceof claim 1, wherein at least one of the first and second substratelayers comprises a plurality of apertures disposed therethrough, theapertures being positioned to be in fluid communication with the atleast first channel when the first and second substrate layers arebonded together.
 8. A microfluidic device comprising at least a firstmicroscale channel having three interior surfaces defined by a firstsubstrate and a fourth interior surface defined by a second substrate,the fourth interior surface comprising 1/4 or less of a total interiorsurface area of the channel.
 9. The microfluidic device of claim 8,wherein first substrate comprises a first groove fabricated into asurface thereof, the groove defining the three interior surfaces of thechannel.
 10. The microfluidic device of claim 8, wherein the firstchannel is from about 20 μm to about 200 μm deep.
 11. The microfluidicdevice of claim 8, wherein the first channel is from about 5 μm to about50 μm wide.